Compound K14 inhibits bacterial killing and protease activity in Dictyostelium discoideum phagosomes

Phagocytic cells of the mammalian innate immune system play a critical role in protecting the body from bacterial infections. The multiple facets of this encounter (chemotaxis, phagocytosis, destruction, evasion and pathogenicity) are largely recapitulated in the phagocytic amoeba Dictyostelium discoideum. Here we identified a new chemical compound (K14; ZINC19168591) which inhibited intracellular destruction of ingested K. pneumoniae in D. discoideum cells. Concomitantly, K14 reduced proteolytic activity in D. discoideum phagosomes. In kil1 KO cells, K14 lost its ability to inhibit phagosomal proteolysis and to inhibit intra-phagosomal bacterial destruction, suggesting that K14 inhibits a Kil1-dependent protease involved in bacterial destruction. These observations stress the key role that proteases play in bacterial destruction. They also reveal an unsuspected link between Kil1 and phagosomal proteases. K14 can be used in the future as a tool to probe the role of different proteases in phagosomal physiology and in the destruction of ingested bacteria.


Introduction
Phagocytic cells ingest, kill, and destroy microorganisms.In mammals, professional phagocytic cells (neutrophils and macrophages) play a key role in protecting the body by eliminating infectious microorganisms [1].In amoebae similar mechanisms are at play [2,3], although the aim is rather to allow amoebae to feed on ingested microorganisms.Intracellular destruction of bacteria is a complex and still incompletely understood process.In mammals, toxic ions, reactive oxygen species, lysosomal enzymes and antibacterial peptides are thought to contribute to the killing and destruction of bacteria in phagosomes [4].However, the relative importance and specificity of each of these mechanisms remains uncertain.The amoeba Dictyostelium discoideum has previously been used as a model phagocytic cell to study how phagocytic cells ingest, kill, and destroy bacteria, and how pathogenic bacteria escape killing by phagocytic cells.It was shown that when a bacterium is ingested by a D. discoideum cell, it is first killed in phagosomes (i.e. it loses the ability to grow), then the bacterial membrane is permeabilized and intrabacterial constituents are destroyed by cellular mechanisms [5].We also identified two key gene products in the bacterial destruction process, named Kil1 [6] and Kil2 [7].Genetic inactivation of Kil1 or Kil2 leads to a strong defect in bacterial intra-phagosomal killing and destruction.Kil1 is a Golgi sulfotransferase participating in the maturation of lysosomal enzymes.Kil2 is a P-type ATPase pumping magnesium ions into the lysosomal/phagosomal lumen.In kil2 KO cells, protease activity in phagosomes is strongly reduced [7], but this is not the case in kil1 KO cells [8].
In previous work we searched for compounds that stimulate destruction of bacteria in phagosomes [9].For this, we used as a tester strain phg1A KO D. discoideum cells, which grow very poorly on a lawn of K. pneumoniae because they fail to destroy and digest them efficiently [8].Phg1A was originally identified as a protein necessary for efficient adhesion, the first step of the phagocytic process [10].It was later found that Phg1A is a protein involved in the maintenance of Golgi integrity [11].Loss of Phg1A prevents transport of the SibA adhesion molecule to the cell surface, causing a loss of cellular adhesion [12].It also prevents proper Golgi localization of Kil1, and this causes a defect in phagosomal destruction of bacteria in phagosomes, similar to the phenotype observed in kil1 KO cells [6].It was expected that some compounds stimulating intracellular destruction of ingested K. pneumoniae would restore growth of phg1A KO cells on K. pneumoniae.Indeed, three compounds (K1-K3), were identified, which stimulated the ability of phg1A KO cells to feed upon K. pneumoniae bacteria [9].The K2 compound (5-ethyl-2'-deoxyuridine) was further shown to act primarily on K. pneumoniae, rendering the bacteria more susceptible to intracellular destruction [9].In this initial screening we did not identify compounds which would act primarily on D. dictyostelium cells by modulating intraphagosomal killing mechanisms.
Here we used a similar strategy to screen additional libraries of chemical compounds.We report the identification and characterization of several new compounds affecting the interaction between D. discoideum and K. pneumoniae.Among them, K14 acts on D. discoideum cells by modifying the physiology of the phagocytic pathway.

Cell and reagents
All D. discoideum cells used in this study were derived from the parental DH1-10 strain [10], referred to as wild-type (WT) for simplicity.D. discoideum mutants used in this study (phg1A KO, kil1 KO, kil2 KO, far1 KO, lrrkA KO, fspa KO and alyL KO were described previously and they are listed in Table 1.D. discoideum cells were grown in HL5 medium [13] at 21˚C. The K. pneumoniae strain used in this study is a non-pathogenic laboratory strain, recently sequenced and named KpGE [18].For all experiments it was grown in LB (lysogeny broth) at 37˚C for 16h.
We used for screening a previously described collection of 8'460 chemical compounds, composed of 6'000 commercially available Maybridge compounds, and from targeted libraries potentially enriched in kinase inhibitors (1200 compounds from Prokinase), in bacterial virulence inhibitors (1'260 compounds from HD-PBL) [19].All compounds were dissolved in DMSO at a concentration of 10mM and aliquots were stored at -20˚C.Aliquots were thawed just before each experiment and immediately thrown away after use.The identification of compounds restoring growth of phg1A KO D. discoideum cells on K. pneumoniae bacteria was done exactly as described previously [9].In each well, growth of D. discoideum cells on a lawn of K. pneumoniae was scored as positive when D. discoideum cleared bacteria on at least 10% of the surface.

Intracellular destruction of bacteria
Intracellular destruction of ingested K. pneumoniae was assessed as described previously [14].Briefly, GFP-expressing K. pneumoniae were washed twice in phosphate buffer (PB)-sorbitol (2mM Na 2 HPO4, 14.7mM KH 2 PO 4 , 100mM sorbitol, pH 6.0) then resuspended in 1 mL PBsorbitol.The suspension was diluted 200 times in PB-sorbitol, and 150 μL of this dilution of bacteria was added on a glass slide (μ-slide 8 well glass bottom, Ibidi GmbH).700'000 D. discoideum cells were washed twice in PB-sorbitol, resuspended in 300 μL of PB-sorbitol, and 100 μL (230'000 cells) deposited in each well.When indicated, the K14 compound was added to the well at the indicated final concentration.Alternatively, DMSO alone (0.3%) was added to the well.The cells were allowed to settle for 5 min, then imaged every 30 sec for 2 h (Nikon Eclipse Ti2).At each time point, one picture (phase contrast and GFP fluorescence) was taken in five successive focal planes (step size 3 μm) to image the whole cell volume.ImageJ was used to analyze movies.Survival analysis of phagocytosed fluorescent bacteria was computed using the Kaplan-Meier estimator.Statistical analysis was done using GraphPad Prism (V8.1.0).Since this assay measures disappearance of bacterial fluorescence in phagosome, it is referred to as bacterial destruction [5].

Intra-phagosomal pH and proteolysis
The proteolytic activity in phasosomes was measured as already described [14] using proteolysis-sensitive bi-fluorescent beads [20].Three-micrometer carboxylated silica beads (Kisker Biotech; PSI-3.0COOH) were coupled with both a proteolysis-sensitive probe (DQ™ Green BSA; Thermo Fisher D12050) and a proteolysis-insensitive probe (Alexa 594 succinimidyl ester; Thermo Fisher A20004).D. discoideum cells were washed with PB-sorbitol and incubated with the beads on a glass slide (μ-slide 8 well glass bottom, Ibidi GmbH), in the presence of 30 μM K14 or 0.3% DMSO.The cells were first allowed to settle for 5 min, then imaged every 75 sec for 3 h (Nikon Eclipse Ti2).At each time point, one picture (phase contrast, GFP and m-Cherry fluorescence) was taken in five successive focal planes (step size 3 μm) to image the whole cell volume.ImageJ was used to analyze movies and to quantify the fluorescence associated with the beads.The phagosomal pH was assessed following the same procedure, using bi-fluorescent beads coupled to a pH-sensitive probe (FITC) and a pH-insensitive probe (Alexa 594), as previously described [14].The FITC/Alexa 594 ratio decreases when beads are exposed to the acidic pH of phagosomes.

Chemical alteration of the interaction of phagocytic amoebae with K. pneumoniae bacteria
To identify chemical compounds which modify the interaction between D. discoideum amoebae and K. pneumoniae bacteria, we grew bacteria and amoebae in the presence of a collection of chemical compounds and identified compounds which altered the ability of amoebae to feed upon bacteria.More precisely, K. pneumoniae bacteria were deposited on nutrient agar (SM medium) in the presence of 30μM of each tested chemical compound, then 10'000 D. discoideum phg1A KO cells were added in the center of the well and allowed to grow at 21˚C for 10 days (Fig 1A).In these pictures, the bacterial lawn appears black, the area from which bacteria were cleared by amoebae appear white (Fig 1B).Within cleared areas, D. discoideum cells are starved and undergo aggregation and multicellular development accounting for the small black dots present in these areas.Because they destroy ingested bacteria inefficiently, phg1A KO cells grew very inefficiently on a lawn of K. pneumoniae.Several libraries of chemical compounds were screened with this assay, for a total of 8'460 compounds (S1 Table in S1 File).After re-testing, 11 new hits were found to restore growth of phg1A KO cells on a lawn of K. pneumoniae and designated K4 to K14 (S2 Table in S1 File).All compounds were reordered except K5 which was no longer available.Their effect was then retested in a more quantitative manner, depositing on a lawn of K. pneumoniae an increasing number of phg1A KO D. discoideum cells, from 1'000 to 30'000 (Fig 1B ), and scoring the effect of compounds on D.

K14 slows down intracellular destruction of bacteria in a Kil1-dependent manner
The fact that K14 modifies the ability of D. discoideum to feed upon bacteria predicts that it affects the interaction between bacteria and D. discoideum cells.K14 may however act potentially on a wide variety of targets either in the bacteria or in the D. discoideum cells.To delineate better the mode of action of K14, we tested its ability to modify the kinetics of K. pneumoniae bacteria destruction in phagosomes.For this, D. discoideum cells were incubated in the presence of GFP-expressing K. pneumoniae and imaged every 30 seconds.Bacteria were ingested, and the disappearance of their fluorescence indicated the time when they were destroyed in the phagosome (Fig 2A).In WT D. discoideum cells, ingested K. pneumoniae were rapidly destroyed.However, K14 slowed down markedly intracellular destruction of ingested bacteria in WT D. discoideum phagosomes (Fig 2B).This effect was visible in multiple experiments and was statistically significant (Fig 2C).
In principle, if K14 inhibits a gene product necessary for efficient destruction (e.g.Kil2), its effect should be lost in the corresponding knockout strain (e.g.kil2 KO strain).To better delineate the molecular process inhibited by K14, we tested the effect of K14 on a collection of D. discoideum mutants known to exhibit slow intracellular bacterial destruction.K14 inhibited bacterial destruction in far1 KO, lrrkA KO, kil2 KO, alyL KO and fspA KO cells (Fig 3A -3E), suggesting that K14 inhibits a cellular process still functional in these mutant strains.On the contrary, in phg1A KO cells bacterial destruction was not slowed down by K14, but rather significantly accelerated (Fig 3F).Previous experiments have shown that phg1A KO cells destroy poorly ingested bacteria largely because they are depleted in the Kil1 protein [8].Remarkably, like in phg1A KO cells, in kil1 KO cells K14 did not slow down bacterial destruction but rather accelerated it (Fig 3G).These observations indicate that the inhibitory effect of K14 is exerted on a target which is inactive in phg1A and kil1 KO cells.Together these results indicate that K14 inhibits intracellular destruction of bacteria in a Phg1/Kil1-dependent manner.In the absence of this dominant Kil1-dependent inhibition, a weaker stimulatory effect of K14 is revealed.This weaker effect presumably accounts for the fact that K14 stimulates growth of phg1A KO cells on K. pneumoniae bacteria.
As detailed above, K14 could inhibit bacterial destruction by inhibiting Phg1, Kil1, or one or several gene products dependent on Kil1 (e.g.proteins sulfated by Kil1).If K14 inhibited Phg1, it should induce a phenotype similar to that of phg1A KO cells, i.e. inhibit bacterial destruction but also cellular adhesion and phagocytosis [10].K14 did not inhibit phagocytosis (S2 Fig in S1 File), indicating that the primary target of K14 is not Phg1 itself.To test whether K14 inhibited Kil1 itself, we tested whether sulfation of proteins was modified by K14 in WT or kil1 KO cells.For this we used an antibody against sulfated antigens (ABCD_AJ514) to assess the presence of sulfated proteins in cell lysates.As expected, many sulfated proteins were detected in WT cells, and virtually none in kil1 KO cells (Fig 4).WT or kil1 KO cells were treated with K14 for either 1 h or 16 h (overnight), and this did not modify the number of sulfated proteins detected in cellular lysates, nor the intensity of the observed signal (Fig 4).These results indicate that K14 does not act by directly inhibiting Kil1 sulfation activity, but rather by interfering with a Kil1-dependent process, most likely inhibiting one or several Kil1-dependent lysosomal enzyme.This interpretation is also coherent with the fact that a treatment with K14 inhibits bacterial destruction much more efficiently than loss of Kil1 (in kil1 KO cells).
All the experiments described above were carried out using a high concentration of K14 (30μM).We next tested the effect of decreasing K14 concentrations.K14 inhibited significantly bacterial destruction in WT cells at a concentration of 1μM, and the inhibition was more prominent at higher concentrations (Fig 5A and 5B).Similarly, K14 stimulated significantly destruction of K. pneumoniae in kil1 KO cells at a concentration of 1μM and the stimulatory effect increased at higher concentrations (Fig 5C and 5D).These observations confirm that both the inhibitory and stimulatory effects of K14 are dose-dependent as would be expected from a classical small molecule inhibitor/activator.

K14 inhibits phagosomal proteolysis in WT but not in kil1 KO cells
The fact that K14 exhibits a different effect on different D. discoideum mutant strains suggests that it acts on the D. discoideum cells rather than on the bacteria, although other more complex interpretations are also possible.More precisely, K14 may modify the physiology of the phagocytic pathway, since it alters intraphagosomal destruction of bacteria.To test this hypothesis  We then measured the proteolytic activity in phagosomes of cells exposed to K14.For this, we used silica beads coupled to two fluorophores: DQ Green coupled to BSA and Alexa 594 [20].The fluorescence of DQ Green is quenched when it is coupled to BSA, but it increases sharply when proteolysis of BSA releases DQ Green from the beads.On the contrary, Alexa 594 provides a constant signal, insensitive to phagosomal proteolysis.As previously reported [14], proteolysis is detectable within minutes following ingestion of beads (Fig 7A).The released DQ Green fluorescence increases steadily and reaches a plateau after approximately 30 minutes (Fig 7B).In WT cells, addition of K14 significantly inhibited the proteolysis (Fig 7B and 7D).On the contrary, in kil1 KO cells, K14 did not inhibit proteolysis (Fig 7C and 7D).These results suggest that K14 inhibits destruction of K. pneumoniae bacteria in WT cells, at least in part, by inhibiting the activity of proteases in phagosomes.On the contrary, in kil1 KO cells, K14 does not inhibit proteolysis, and fails to inhibit bacterial destruction.

Discussion
In this study, we identified K14 as a compound modulating the intracellular destruction of K. pneumoniae bacteria in D. discoideum phagosomes.In WT cells, K14 inhibits destruction of K. pneumoniae bacteria in phagosomes.Two separate lines of evidence indicate that this effect is primarily due to the action of K14 on cellular physiology.First K14 inhibits destruction of ingested bacteria in the phagosomes of WT cells, but not in phg1A KO or kil1 KO cells.This observation suggests that K14 acts on a D. discoideum cellular mechanism which is inactive in kil1 KO and in phg1A KO cells.K14 does not modify protein sulfation by Kil1, but probably acts on a lysosomal enzyme which requires sulfation by Kil1 to be active.Second, in the absence of bacteria, the enzymatic activity of proteases in WT D. discoideum phagosomes is inhibited by K14.Remarkably, in kil1 KO cells, K14 does not inhibit proteolytic activity in phagosomes, as would be expected if K14 inhibits a Kil1-dependent phagosomal protease.This observation suggests that K14 inactivates a phagosomal protease which is inactive or depleted from phagosomes in kil1 KO cells.The inhibition of bacterial destruction and of phagosomal protease activity is observed within minutes of exposure to K14.The effect of K14 may thus be achieved either by modifying the composition of newly forming phagosomes (e.g. by inhibiting the phagosomal targeting of a critical protease), or by directly inhibiting the activity of a protease.If phagosomes of WT cells contain a protease inhibited by K14, and this protease is absent or inactive in kil1 KO cells, one would expect the total proteolytic activity to be reduced in kil1 KO cells.Previous analysis, as well as this study indicate on the contrary that the total proteolytic activity in kil1 KO cells is quantitatively similar to that in WT cells.This suggests that in kil1 KO cells the loss of activity of a K14-dependent protease is compensated by the up-regulation of some other K14-insensitive protease.This compensatory effect explains why previous studies failed to detect an alteration of phagocytic proteases in kil1 KO cells.It should be emphasized that the tools that we used in this study to detect phagosomal protease activity detect any protease capable of digesting BSA.More specific substrates may be necessary to detect the modification of protease activities in kil1 KO cells compared to WT cells.
In phg1A KO cells, as well as in kil1 KO cells, K14 does not inhibit intracellular destruction of bacteria, and on the contrary it stimulates it.It is likely that this stimulation of bacterial destruction accounts for the increased growth of phg1A KO cells on a lawn of K. pneumoniae bacteria, the phenotype initially used to identify K14.In retrospect, it is curious to note that the K14 compound was not identified based on its primary inhibitory effect on WT D. discoideum cells, but rather on a weaker stimulatory effect observed only in phg1A KO and kil1 KO mutant cells.While it may be interesting to investigate the molecular nature of this secondary stimulatory effect of K14, this effect is quantitatively less pronounced than the primary inhibitory effect of K14.
K14 is the second chemical compound identified in our studies that modulates the physiology of phagosomes and destruction of bacteria (schematized in Fig 8).Indeed, previous studies showed that folate, a bacterially secreted compound, stimulates destruction of K. pneumoniae in phagosomes.This stimulatory effect was lost in mutants where folate sensing was lost: far1 https://doi.org/10.1371/journal.pone.0309327.g007KO (the surface receptor for folate), lrrkA KO (a cytosolic kinase), and kil2 KO (an ion pump believed to pump magnesium into the phagosome lumen).In these mutants, a decrease in phagosomal proteolytic activity accompanied, and probably caused the slower destruction of K. pneumoniae bacteria [14].Folate was proposed to stimulate the Far1-LrrkA-Kil2 pathway, inducing an increase in phagosomal magnesium and thus stimulating phagosomal proteases and the destruction of ingested bacteria.In the current study, K14 slows down bacterial destruction by inhibiting the activity or the delivery of a Kil1-dependent protease in phagosomes.These new observations stress further the importance of proteolytic activity in D. discoideum phagosomes to achieve efficient destruction of bacteria.To date, no individual phagosomal protease has been identified that is required for efficient destruction of bacteria in phagosomes.A partially purified extract of D. discoideum exhibiting bacteriolytic activity contained 15 proteases [23] (tpp1B, C, E, F; ctsB, D, Z; cprA, D, E, G; Q54CF7, Q54MN6, Q54VR1, Q54F16/p34), at least two of which (ctsD, p34) were previously shown to be present in the endocytic/phagocytic pathway [24,25].Our results may help in the identification of a phagosomal protease involved in the destruction of K. pneumoniae bacteria: this protease(s) should be inactive (or absent from phagosomes) in kil1 KO cells, stimulated by magnesium ions, and inhibited (or depleted from phagosomes) in cells exposed to K14.

Fig 1 .
Fig 1. Selection of compounds restoring growth of phg1A KO D. discoideum cells on K. pneumoniae. A. D. discoideum cells were deposited on a lawn of K. pneumoniae bacteria.After 10 days, D. discoideum cells formed a phagocytic plaque if they were capable of feeding upon the bacteria.B. Selected compounds (here K14) increased the ability of phg1a KO cells to create phagocytic plaques compared with the negative control (DMSO), scale bar: 2 mm.C. The effect of each compound was scored from 4 (efficient growth of 1'000 phg1A KO cells) to 0 (no visible growth of 30'000 cells) and the score of the negative control substracted.In the example shown in B the scores would be 1 for DMSO and 3 for K14, resulting in a growth score of 3-1 = 2 for K14.Mean ± SEM; *: p<0.05;Kruskal-Wallis test, Dunn's test.N = 7 independent experiments.D. Chemical structure of the K14 compound.https://doi.org/10.1371/journal.pone.0309327.g001

Fig 2 .
Fig 2. K14 inhibits the destruction of ingested K. pneumoniae in WT D. discoideum phagosomes. A. Time-lapse images showing a fluorescent K. pneumoniae (arrowhead) ingested by a WT D. discoideum.Extinction of the fluorescence was observed after 10 min in the upper movie (DMSO control).In the presence of K14, the fluorescence of the bacterium disappeared after 57 min (scale bar: 10 μm).B. Kaplan-Meier survival curves of ingested bacteria were calculated for experiments performed in the absence (DMSO) or presence of K14 (30μM) (n = 150 phagocytic events, N = 5 independent experiments).C. For each independent experiment, the area under each survival curve (AUC) was determined.The difference between the AUC for K14-treated and untreated cells was determined.A positive value indicated an experiment where intracellular destruction was slower than in the control.K14 slowed down significantly the intracellular destruction of K. pneumoniae by WT D. discoideum cells (mean ± SEM; *: p<0.05;Mann-Whitney test.N = 5).https://doi.org/10.1371/journal.pone.0309327.g002

Fig 4 .
Fig 4. The sulfation of proteins is not affected by a K14 treatment.The AJ514 scFv antibodies recognizes the sulfated common antigen 1 and was used to detect sulfated proteins by western blot.Many proteins were detected in lysates from WT D. discoideum cells but not in the sulfation-defective kil1 KO mutant.Incubation of the cells with K14 (30μM) for 1h or overnight prior to lysis did not modify the sulfation pattern observed.The original uncropped gel is shown in S4 Fig in S1 File.https://doi.org/10.1371/journal.pone.0309327.g004

Fig 5 .
Fig 5. K14 inhibits intracellular destruction of K. pneumoniae in WT cells and stimulates it in kil1 KO cells at similar concentrations.A. The effect of K14 on the intracellular destruction of K. pneumoniae by WT D. discoideum cells was determined as described in the legend to Fig 2. B. K14 inhibited significantly bacterial destruction at concentrations of 1μM and higher (mean ± SEM; *: p<0.05;Mann-Whitney test, N = 5 independent experiments).C. In kil1 KO cells, intracellular destruction of ingested bacteria was much slower than in WT cells, and it was stimulated by K14 at a concentration of 1μM and higher.D. Analysis of 7 independent experiments revealed that the stimulatory effect of K14 was statistically significant (mean ± SEM; *: p<0.05;Mann-Whitney test, N = 7).https://doi.org/10.1371/journal.pone.0309327.g005

Fig 6 .
Fig 6.K14 does not alter the acidification of phagosomes. A. Acidification of phagosomes was assessed by measuring the fluorescence of ingested silica beads coupled to two fluorophores, one quenched by pH (FITC, green), one not (Alexa 594, red).Shortly after ingestion (t = 0), FITC fluorescence was quenched by the acidic phagosomal pH and the fluorescence of the beads switched from yellow (green+red) to red.For each picture an inset shows the fluorescence of the bead in the FITC channel.Scale bar: 10μm.B. Acidification of phagosomes proceeded with the same kinetics in WT cells treated with K14 (30μM) or not (DMSO).C. Similarly, in kil1 KO cells, acidification of phagosomes was indistinguishable from acidification in WT cells and was not inhibited by K14.(mean ± SEM; WT: N = 13; kil1 KO: N = 11).https://doi.org/10.1371/journal.pone.0309327.g006

Fig 7 .
Fig 7. K14 inhibits proteolytic activity in phagosomes in WT but not in kil1 KO cells.A. Proteolytic activity in phagosomes was assessed by measuring the fluorescence of ingested silica beads coupled to BSA-DQ Green.Following ingestion (t = 0), digestion of BSA and release of DQ Green leads to a marked increase in fluorescence.Beads were also coupled to a proteolysis-insensitive fluorophore to allow continuous observation (Alexa 594, red).Scale bar: 10μm.B. Release of fluorescent DQ-Green in phagosomes was measured as a function of time to assess the proteolytic activity in maturing phagosomes.Phagosomal proteolysis was inhibited in WT D. discoideum cells treated with K14 (30μM).(N = 5 independent experiments, n = 75 ingested beads).C. Proteolytic activity in phagosomes was not modified in kil1 KO cells exposed to K14 compared with DMSO.(N = 7 independent experiments, n = 105 ingested beads).D. To evaluate the statistical validity of these results, the results of individual experiments were compared (mean ± SEM; *: p<0.05;Mann-Whitney test).

Fig 8 .
Fig 8. Effects of folate and K14 on phagosomal physiology.The primary effect of folate is to stimulate the Far1-LrrkA-Kil2 pathway, ultimately increasing magnesium concentration in phagosomes.Magnesium stimulates phagosomal proteases necessary for destruction of K. pneumoniae (Kp) bacteria.The primary effect of K14 is to inhibit Kil1-dependent proteases involved in bacterial destruction.Phagosomal proteases involved in bacterial destruction are stimulated by magnesium, inhibited by K14, and by genetic inactivation of Kil1.https://doi.org/10.1371/journal.pone.0309327.g008